The correct execution of a developmental program requires that individual events proceed in an orderly fashion. Differentiating cells have to integrate processes such as DNA replication, cell division, and changes in morphology. The long-term goal of this project is to understand how cell division and cell differentiation are regulated and integrated in the bacterium Caulobacter crescentus. Each cell division is asymmetric and produces two different cell types: a motile swarmer cell and a sessile stalked cell. Only the stalked cell is competent to replicate DNA and divide. The asymmetric predivisional cell has a flagellum at one pole and a stalk at the opposite pole. The establishment of asymmetry prior to cell division is tightly coupled to cell cycle progression by DNA replication and cell division checkpoints. The proposed research has three main objectives. The first objective is to identify the mechanisms responsible for the cell cycle control of cell division. One checkpoint that couples cell division to DNA replication is mediated by the master cell cycle response regulator CtrA. Experiments are proposed to determine how the activity of CtrA is regulated by DNA replication and how the cell sets the stage for the replication checkpoint by degrading cell division proteins at the end of every cell cycle. The second objective is to define the genes and the cell division checkpoint mechanism that couple polar development to cell division. The sigma-54 specific response regulator, TacA, is required for the cell division checkpoint and its mechanism of action will be determined. The TacA-dependent gene(s) involved in checkpoint control will be identified and studied, and the mechanism by which cell division inhibition is transduced to TacA will be determined. The third objective is to investigate the function of the polar organelle development protein, PodJ, in regulating pili and holdfast synthesis, two events that are blocked by the cell division checkpoint. The role of PodJ in the localization of critical regulators of development will be determined and the mechanism of PodJ proteolytic processing, which is coupled to cell division, will be investigated. These studies will lead to a better understanding of the mechanisms that regulate cell differentiation.